JP3181979B2 - Orotic acid derivative-containing composition and functional thin film - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an orotic acid derivative suitable for use as a sensor having high substrate selectivity, a carrier used for transport and extraction of a substrate, a sustained release agent, a reaction field, and the like. The present invention relates to a functional thin film.

[0002]

2. Description of the Related Art A sensor responding to a chemical substance is called a chemical sensor, and includes (1) an ion sensor represented by a pH sensor, (2) a gas sensor, and (3) a biosensor. The ion sensor detects the concentration of the chemical substance contained in the liquid to be measured as a change in the membrane potential of the ion-sensitive membrane. There are a wide variety of gas sensors.For example, semiconductor gas sensors measure the change in electrical resistance when gas molecules are adsorbed, and potentiostatic and galvanic cell type gas sensors use electrochemical redox. Is what you do. In these ion sensors and gas sensors, the physical properties of the sensitive part change depending on the detected object, and the amount of change is extracted as a detection signal. In addition, there are many inorganic molecules to be measured.

On the other hand, detection of organic molecules is required in many fields such as medical examination, fermentation, food industry, bioengineering and the like. As this detecting means, a biosensor such as an enzyme sensor is used. In this biosensor, a biological substance capable of selectively recognizing a specific substance is used, and a change in a substance included in a chemical reaction with the substance to be measured is extracted as a detection signal.

That is, in a conventional biosensor, a biological substance which reacts specifically with a substance to be detected is selected, and products such as oxygen and hydrogen peroxide which can be detected by an ion sensor or a gas sensor are provided. Then, in combination with an ion sensor or a gas sensor, it becomes possible to detect various kinds of organic molecules, and it is also possible to measure a trace amount.

[0005]

However, in the conventionally used biosensor, the sensitive part and the signal generating part are separately configured in terms of their functions.
It has a complicated structure. In addition, a sensor using an immobilized enzyme or the like has a drawback that the activity of the enzyme changes with time and the temperature used is limited to a physiological temperature determined by the type of the enzyme.

In this regard, there is a demand for the development of an organic substance sensor having a sensing response mechanism similar to an ion sensor or a gas sensor for an inorganic compound, and some studies are being conducted on a trial basis. For example, Okahata is Poly
mer Preprints. Japan Vol. 3
7, No. 10th 3309-3311 (1988)
Using a crystal oscillator and a porous polymer membrane covered with a bilayer membrane, we found that when various aqueous alcohols were adsorbed on the bilayer membrane, the weight, membrane potential, membrane resistance, etc., changed, respectively. It reports that it can be used. The molecular identification here is performed by distributing the hydrophobic molecules to the hydrophobic layer of the membrane.

Further, Odashima et al. Have reported a catechol-type sensitive sensor using a liquid membrane containing a fat-soluble macrocyclic polyamine as a molecular recognition element [The 3rd Symposium on Biological Function-Related Chemistry, pp. 165-167] (1988)]. In this report, it has been found that catechols, which are neutral molecules, exhibit potential response, and suggest that proton ejection by hydrogen bond interaction between host and guest is a mechanism of potential response.

However, the sensor reported by Okahata et al.
There is a disadvantage that the target compound is limited to fat-soluble compounds. On the other hand, the sensor of Odashima et al. Does not clarify the cause of the response and is of a liquid film type, so that the amount of physical properties that can be measured is limited to a potential change. In addition, since the host-guest interaction used is of a homogeneous solution type, it is necessary to synthesize a molecule that binds an analyte in a homogeneous solution type. Therefore, there is a disadvantage that the selectivity, sensitivity, and operability are reduced.

Therefore, an object of the present invention is to provide a substance useful as a novel organic substance sensor corresponding to the above-described types such as the ion sensor and the gas sensor.
In addition to a sensor, a new orotic acid derivative used as a reaction field for providing a carrier, a sustained-release agent, and a reaction catalyst used for transporting and extracting a substrate, and a film formed using the derivative. It is intended to provide a functional thin film.

[0010]

The orotic acid derivative-containing composition according to the present invention, in order to achieve the object, comprises a nucleoside, a nucleobase, a water-soluble peptide or a water-soluble polymer in the form of a compound or an inclusion crystal. The carboxyl group or imino group of orotic acid has at least one carbon atom
It is characterized by containing an orotic acid derivative substituted with a long-chain alkyl group, a long-chain fluoroalkyl group or a derivative thereof of 0 to 22.

The orotic acid derivative can be formed into a functional thin film by an LB method, a casting method, a dispersion method, or the like. In the formed thin film, imide groups are oriented on the surface due to organization of molecules. Further, the thin film can be immobilized on the solid surface by physical adsorption or chemical adsorption using a silane coupling material or the like.

[0012]

The orotic acid derivative according to the present invention is obtained by introducing at least one alkyl or fluoroalkyl having 10 to 22 carbon atoms or a derivative thereof into a carboxyl group or an imino group of orotic acid having the following structure. is there. When the orotic acid derivative is formed into a film, an interesting thin film in which the direction of the imide group as a hydrophilic group is controlled can be obtained.

[0013]

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That is, by introducing an alkyl group into orotic acid, the orientation of the imide group in the prepared thin film is controlled, and a specific substrate is bound using the site. The orotic acid derivative can be used in various solvents including water, and is produced into a functional thin film having high density and high sensitivity and selectivity.

The orotic acid derivative is obtained by subjecting a carboxyl group of orotic acid having the above structural formula to a treatment such as amidation, imidation, or esterification to obtain at least one ore having 10 to 10 carbon atoms.
It is obtained by introducing 22 long-chain alkyl groups. Alternatively, an orinoic acid derivative is synthesized by introducing a long-chain alkyl group having 10 to 22 carbon atoms by alkylating an imino group. The following equation shows an example of the synthesis process.

[0016]

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The synthesized orotic acid derivative has a self-organizing property. For example, when an orotic acid derivative is developed at an aqueous interface, an imide group, which is a hydrophilic group, is oriented at the interface, and a hydrophobic group R is arranged in an opposite direction. Furthermore, when the thin films developed at the interface are accumulated on the substrate, an LB film maintaining this arrangement can be obtained. At this time, even when mixed with other film-forming compounds such as lecithin and long-chain dialkylammonium, an LB film that maintains the properties of the orotic acid derivative can be obtained.

Regardless of the developing method, when a solution obtained by dissolving an orotic acid derivative in an organic solvent such as acetone or chloroform is cast on a substrate such as a glass plate and formed into a film by a natural drying method, the film may be further appropriately formed. Similar self-assembly of molecules can be performed by dispersing in water to form liposomes. The prepared thin film is stabilized when fixed on the surface of the solid substrate by physical adsorption or chemical adsorption.

The oriented imide groups selectively bind the substrate by forming hydrogen bonds. For example, when a monomolecular membrane is prepared using an aqueous liquid to which various nucleosides, nucleobase derivatives, water-soluble peptides, water-soluble polymers, and the like are added, the film-forming behavior exhibits different substrate selectivity depending on the substrate. .

The imide group has two hydrogen bonding properties.
It has CO and one = NH, and a combination of many hydrogen bonds can be provided by these three hydrogen bonding functional groups and a plurality of functional groups of a substrate such as a nucleoside.
This combination changes the strength of the interaction that occurs between the two compounds, and changes the behavior during film formation according to the strength and the molecular size of the compound serving as the substrate. The functional thin film of the present invention is used as a sensor in a wide range of fields utilizing this property.

The orotic acid derivative of the present invention forms an inclusion compound selectively with a substrate when crystallized from a suitable solvent containing the substrate. The substrate incorporated in the orotic acid derivative can be dissociated from the thin film or the inclusion crystal by solvent extraction, pH adjustment, heat treatment, or the like, and taken out. Therefore, carriers used for transport and extraction of substrates,
It can also be used as a carrier for slowly releasing a substrate, such as a sustained release agent such as a drug in the field of pharmaceuticals, and as a reaction field for providing a reaction catalyst. Further, since the binding strength of various nucleosides to the substrate and the like are different, the release rate between the substrate and the substrate can be controlled.

[0022]

EXAMPLE 1 200 g of octadecyl alcohol (I) and 2.0 g of sodium hydride were placed in a 500 ml three-necked flask and kept at 70.degree. To this, epichlorohydrin (7 g) was gradually added dropwise over 30 minutes while stirring. After the reaction,
Excess octadecyl alcohol was distilled off under reduced pressure, and recrystallized from chloroform to obtain 30.0 g of dioctadecylglyceryl diether (II). The reaction at this time is shown in the following equation.

[0023]

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2.0 g of orotic acid (III) was suspended in dry benzene, and 5.0 g of thionyl chloride was gradually added thereto at room temperature, and 0.5 ml of anhydrous DMF was further added. The mixture was refluxed for 6 hours with stirring, cooled to room temperature, and then filtered. The obtained reaction product was washed with anhydrous benzene to obtain 2.1 g of orotic acid (IV). The reaction at this time is shown in the following equation.

[0025]

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0.7 g of the obtained dioctadecylglyceryl diether (II) and 0.05 g of sodium hydride were added to 1
Anhydrous THF in a 00 ml three-necked flask
30 ml, and further add 0.1 ml of orotic acid (IV) chloride with stirring.
2 g was added slowly. The mixture was refluxed for one day under stirring, and the reaction solution was separated by filtration. The reaction product obtained by evaporating the solvent was recrystallized from ethanol and benzene to obtain 0.36 g of the target compound (V). The reaction at this time is shown in the following equation. The yield was 42.7%.

[0027]

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Compound (V) has a melting point of 102.4-10.
3.4 ° C., the result of elemental analysis was C: 71.98%,
H: 11.26% and N: 3.67%. This analytical value is a calculated value C: 71.89 obtained from the chemical structural formula.
%, H: 11.24% and N: 3.81%. In addition, the IR value of compound (V) was as follows. 3200, 3080 cm ^-1 (NH) 2914,2
846 cm ^-1 (CH ₂ ) 1673, 1492, 1465,
1261,1125cm ^-1

¹ H NMR shows that δ 8.56 (br
s, 1H, 3-NH), 8.43 (br s, 1H,
1-NH), 6.41 (s, 1H, CH), 5.33
(Tt, J = 3.7 Hz, 1H, OCHCH ₂ ), 3.62
(D, J = 4, 4H, CHCH ₂ O), 3.43 (m,
4H, OCH ₂ ), 1.59, 1.53 and 1.24
(M, 64H, alkyl tail), 0.87
(T, J = 5, 6H, CH ₃ ).

Example 2: Production of monomolecular film Orotic acid derivative (V) produced in Example 1 6.4m
g of a mixed solvent of 9 parts of chloroform and 1 part of ethanol.
Then, 70 μl of the solution dissolved in ml was spread on the water surface using an LB film production apparatus kept at 20 ° C. And 0.2m
A monolayer was formed by compressing at a speed of m / sec. Keep the surface pressure during compression at a constant value of 20 mN / m,
The LB film was produced by moving the substrate up and down at a constant speed of 20 mm / min and accumulating the monomolecular film formed on the water surface on the substrate.

Various materials such as a glass plate, quartz, a polymer film, graphite, silver, and gold were used as the substrate. The resulting LB film had a Y-type structure as a result of the cumulative behavior regardless of which substrate was used.

Example 3: Characteristics as a sensor (1) When a monomolecular film is prepared as in Example 2 using an aqueous phase containing a substance serving as a substrate for a monomolecular film constituent molecule,
By monitoring the surface pressure and the molecular occupation area, the effect of the substrate on the monolayer can be investigated.

For example, the orotic acid derivative (V) obtained in Example 1 is spread on the surface of an aqueous solution of adenine having various concentrations at 20 ° C., and a monomolecular film is formed at a compression rate of 0.2 mm / sec. The measurement results were collected at concentrations of 1 mM, 2 mM and 3 mM.
FIG. 1 shows a surface pressure-molecule occupied area curve when an aqueous solution of adenine was used.

As is clear from FIG. 1, the collapse pressure of the orotic acid derivative monolayer varies depending on the concentration of the aqueous adenine solution. Then, the concentration of adenine can be known by measuring the collapse pressure obtained from the surface pressure-molecule occupation area curve.

Similarly, the surface pressure is reduced by using an aqueous thymine solution.
The molecular occupation area curve was measured. At this time, the surface pressure-molecule occupied area curve was consistent with the surface pressure-molecule occupied area curve obtained from pure water without depending on the concentration of the thymine aqueous solution. This indicates that, in an aqueous solution containing both adenine and thymine, the orotic acid derivative thin film has a function of selectively recognizing adenine. Further, using these results, the type of the substrate can be determined from the surface pressure-molecule occupied area curve. Such substrate specificity is presumed to be due to the ease of hydrogen bond interaction between the substrate and the orotic acid derivative, and the type of long-chain alkyl group or long-chain fluoroalkyl group introduced into orotic acid is considered. The sensor is used as a sensor for selectively recognizing various substrates by changing the above.

Example 4: Characteristics as a sensor (2) Orotic acid derivative (V) produced in Example 1 (6.4 m)
g of a mixed solvent of 9 parts of chloroform and 1 part of ethanol.
By dissolving the solution in ml, a developing solution was prepared. Using an LB membrane manufacturing apparatus, 100 μl of this developing solution was spread on the water surface of the aqueous phase containing a substance serving as a substrate for a monomolecular film constituent molecule, as in Example 3. Then, a monomolecular film is formed at a speed of 0.2 mm / sec, and the surface pressure during compression is set to 20 mN / m.
The quartz plate which had been immersed in the aqueous phase in advance was pulled up while maintaining the constant value of. Thus, the monomolecular film formed on the water surface was transferred to the quartz plate, and an LB film composed of one monomolecular film was formed on the surface of the quartz plate.

When the UV spectrum of the obtained LB film was measured, as shown in FIG. 2, the absorbance at 268 nm increased as the concentration of adenine added to the aqueous phase increased. Note that the change in absorbance in FIG. 2 is represented by a difference from the absorbance at 268 nm of the LB film similarly obtained from pure water. Conversely, the adenine concentration in the aqueous phase can be known by measuring the absorbance at 268 nm of the prepared LB film using the relationship between the adenine concentration and the change in absorbance.

Example 5: Immobilization of substrate 6.4 mg of orotic acid derivative (V) obtained in Example 1
In a mixed solvent of 9 parts of chloroform and 1 part of ethanol 10 m
and dissolved at 20 ° C. using an LB film manufacturing apparatus.
On an aqueous solution of adenine and thymine (3 mM)
Developed 00 μl. The surface pressure during compression was maintained at a constant value of 20 mN / m, and the gold vapor deposition plate was moved up and down at a constant speed of 20 mm / min, and immersion and pulling were repeated three times. As a result, six monolayers formed on the water surface are accumulated L
A B film was obtained.

FT-IR (RAS method) of the obtained LB film
When the spectrum was measured, as shown in FIG. 3, the spectrum of the LB film obtained from the adenine aqueous solution was 3445 c compared to the spectrum of the LB film obtained from the pure water.
absorption of NH adenine combined with imide group in m ^-1 and 3336cm ^-1 were observed, further 1677Cm ^-1 and 164
At 7 cm ^-1 , a new absorption attributed to the bending vibration of the hydrogen bonding NH of adenine was observed. From this spectral analysis,
It can be seen that adenine is fixed on the obtained LB film.

On the other hand, the spectrum of the LB film obtained from the thymine aqueous solution was almost the same as the spectrum of the LB film obtained from the pure water. This indicates that thymine was not immobilized on the LB film of the orotic acid derivative. This also indicates that the orotic acid derivative exhibits high substrate selectivity.

[0041]

As described above, when the orotic acid derivative of the present invention is used, nucleosides, nucleobase derivatives, water-soluble peptides, ATP, etc. are regularly taken in between the layers in the membrane structure at the molecular level. It becomes possible to produce thin films such as liposomes, LB membranes, monomolecular membranes, cast membranes and the like. In addition, a compound having a self-organizing property alone or an imide group regularly arranged on a thin film by organizing molecules can be used as a sensor for various substances.

Further, the substrate taken into the membrane can be dissociated and removed from the thin film or the inclusion crystal by solvent extraction, pH adjustment, heat treatment and the like. So, substrate transport,
An orotic acid derivative thin film can also be used as a carrier for extraction and storage. In addition, since the physiologically active substance is released effectively and effectively over a long period of time, it is promising as a means for preparing a drug including a sustained release agent. As described above, the orotic acid derivative of the present invention and the functional thin film produced from the derivative are expected to be used in a wide range of fields.

[Brief description of the drawings]

FIG. 1 is a graph showing a surface pressure-molecule occupation area curve of a monomolecular film obtained in Example 3 of the present invention.

FIG. 2 shows 268 of the LB film similarly produced in Example 4.
Graph showing the change in absorbance at nm in relation to the adenine concentration in the aqueous phase

FIG. 3 is FT-I of the LB film similarly obtained in Example 5.
R spectrum

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-1-226876 (JP, A) West German Patent Application Publication 3206197 (DE, A1) Chemistry Letters; (1992) No. 9 p1839-1842 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 239/557 B01D 69/00 G01N 27/327 CA (STN) REGISTRY (STN)

Claims (3)

(57) [Claims] 1. A nucleoside, a nucleobase, a water-soluble peptide.
State or inclusion crystal state
In the state, the carboxyl group or imino group of orotic acid is
At least one long chain alkyl group having 10 to 22 carbon atoms,
Substituted with long-chain fluoroalkyl groups or their derivatives
Orochi characterized by containing orotic acid derivatives
An acid derivative-containing composition. 2. An orotic acid derivative-containing set according to claim 1.
The product is formed into a film by LB method, cast method or dispersion method,
The imide groups were oriented toward the membrane surface by self-assembly
A functional thin film characterized in that: 3. Claim by physical or chemical adsorption
2 that the functional thin film is immobilized on a solid surface
Characteristic functional thin film.